Immunologically based strip test utilizing ionophore membranes

ABSTRACT

A testing apparatus  10  having an absorbent matrix  12 , including a membrane  14  which contains a plurality of counter-ions  16.  Chromionophore (or fluorionophore)s  18  and affinophores  22  compete to carry ions into the membrane  14  and neutralize the charge of the counterions  16.  Biological recognition molecules  42  bind to a portion of the affinophores  22  and prevent them from entering the membrane  14,  thereby allowing more chromionophore (or fluorionophore)s  18  to enter the membrane  14.  The portion of affinophores  22  bound to the biological recognition molecules  42  is inversely proportional to the amount or concentration of analyte  40  occurring within the solution or medium  30.  The result of this is that the color of the membrane-covered matrix changes in a manner related to the concentration of the analyte. One application of this apparatus is a strip test for prediction of ovulation.

[0001] The U.S.. Government has a nonexclusive, nontransferable,irrevocable paid-up license to practice or have practiced this inventionfor or on its behalf as provided for by the terms of Contract No.DAMD17-96-C-6026 awarded bt the U.S. Department of the Army

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to an apparatus and a methodology todetermine the presence and/or concentration of certain metabolites orother substances occurring within a certain medium, such as and withoutlimitation, blood, urine, and saliva. In one embodiment the apparatusand methodology utilizing the principles of the invention is adapted foruse as a birth management tool.

[0004] 2. Background of the Invention

[0005] Many types of “home-based” testing apparatuses exist to determinethe presence and/or concentration of substances such as metabolitesoccurring within some medium. While these prior “home-based” testingapparatuses often adequately determine the presence of some of thesesubstances, such as metabolites, they are not capable of determining theconcentration or, in many cases, even the presence of such targetsubstances when the concentration of these metabolites or othersubstances is less than about 10⁻⁶M. In these cases, relatively complexand costly laboratory tests must be done, usually by one or more trainedtechnicians, in order to ascertain the presence and/or concentration ofthese substances. There is therefore a great need for a technique(hereinafter the term “technique” as used in this Application refers toboth an apparatus and a methodology) to reduce health care cost byproviding a relatively inexpensive and relatively easy to use“home-based” testing apparatus to determine the presence and/orconcentration of certain substances or metabolites. Applicant'sinvention addresses and fulfills this need. Moreover, there is also agreat need for a “real time” diagnostic technique that will providereliable data concerning the presence and/or concentration of certainmetabolites and other substances occurring within a certain medium andwhich will allow an individual to take a prompt action in response tothe diagnostic indications. Applicant's invention provides such a “realtime” diagnostic indication.

[0006] Particularly, Applicant has found that these various needs areparticularly acute in the field of birth control and birth managementand that Applicant's invention, in one embodiment, is particularlysuited for use in birth control and birth management. That is, it hasbeen deduced form a retrospective study of family planning records thatabout 90% of all conceptions occur within about a five day period whichprobably spans the day of the peak concentration of the luteinizinghormone occurring and/or most often sensed within human urine, and moreparticularly, probably spans from about two days before this peak occursuntil about three days after the peak arrives. It is known that duringthis fertile period, sexual intercourse may lead to pregnancy. Hence,many of the current “home-based” birth management techniques utilizesome sort of color changeable paper to notify the users of the pendingfertility period by measuring the concentration of the luteinizinghormone. While somewhat useful, these prior techniques providenotification of the impending ovulation no more than thirty-six hoursprior to the onset of the fertile period. Since the average longevity ofsperm in the vagina is about forty-eight hours, these prior “home-based”tests do not detect the start of the fertility cycle sufficiently earlyto reliably prevent conception and/or to actually “manage” theconception process.

[0007] It is known that the 17-estradiol metabolite,estrone-3-glucuronide (commonly referred to by those of ordinary skillin the art as “E1-g”) reaches about 85% to about 95% of its peak valuein human urine within about 72 hours prior to the onset of ovulation.Hence, the concentration measurements of E1-g in urine, as Applicant hasfound, provides a reliable advance warning of the onset of ovulation,sufficient to prevent conception since the time of warning is longerthat the lifetime of sperm in the vagina and sufficient to allowindividuals to “manage” conception (e.g. actively determine and planwhen conception should begin). Prior “home-based” techniques are notreadily able to measure E1-g within the urine because the gradualmonotonic peak of E1-g requires a more quantitative detection devicethan current tests provide. Moreover, the World Health Organization hasdetermined the “home-based” E1-g birth management systems would be ofgreat utility in managing the worfd's population and would be especiallyuseful in overpopulated and developing countries since birth managementcould be achieved (e.g. birth control could be achieved by abstinenceduring the ovulation period while conception could be more readilyachieved by sexual intercourse during ovulation). Applicant's invention,in one embodiment, provides such an E1-g sensing and/or testingapparatus for use in “home-based” birth management and provides theutility sought in the developing and overpopulated countries.Applicant's invention is therefore an advance in the art of birthmanagement and contains general inventive principles which have a wideuse in many other areas of sensing systems.

SUMMARY OF THE INVENTION

[0008] It is a first objective of the present invention to provide arapid measurement technique for determining the presence and/orconcentration of certain analytes (metabolites and/or other substanceswithin a medium such as urine, blood, and/or saliva) which overcomes thevarious disadvantages of the prior art.

[0009] It is a second object of the present invention to provide a“homebased” technique to determine the presence and/or concentration ofcertain analytes and other substances within a medium.

[0010] It is a third object of the invention to provide a technique todetermine the concentration of the analyte, E1-g, in a medium.

[0011] It is a fourth object of the present invention to provide atechnique which generates a certain color which is indicative of theconcentration of a certain analyte, such as E1-g, within a medium.

[0012] It is a fifth object of the present invention to provide atechnique to provide for “home-based” birth management in a manner whichis superior to that of the prior art.

[0013] It is a sixth object of the present invention to provide atechnique for measuring the concentration and/or occurrence of a certaintarget analyte occurring within a medium by the use of first and secondsubstances which compete for entry into a membrane which is constrainedto remain electrically neutral.

[0014] It is a seventh object of the present invention to provide atechnique utilizing a matrix type strip which is adapted for placementwithin a target medium and which is further adapted to provide a certaincolor indication at a certain portion thereof, indicative of theconcentration of a certain target analyte occurring within the targetmedium.

[0015] It is an eighth object of the present invention to provide atechnique utilizing a continuous strip which is adapted for placementwithin a target medium and which is further adapted to display certaincolor indication whose position along the strip is indicative of theconcentration of a certain target analyte occurring within the targetmedium.

[0016] It is a ninth object of the present invention to provide anaffinophore molecule which is synthesized so as to have a certainbinding affinity for antibody to estrone 3-glucuronide, which allows thebiological recognition molecule to modulate the entry of two competingsubstances within a membrane by an amount which is proportional to theconcentration of a certain third substance within the medium. Ingeneral, an affinophore for use in Applicant's techniques is createdfrom a commercially available ionophore which has been chemically and/orbiologically altered or conjugated with or to a molecule so as to have abinding affinity for a biological recognition molecule which has abinding affinity for the metabolite or other substance whoseconcentration is to be measured. The biological recognition molecule mayalternatively comprise an antibody, a portion of an antibody, abiological receptor for the analyte, a portion of a nucleotide sequencehaving chemical and/or biological affinity for the analyte, or virtuallyany other substance which has some chemical and/or biological bindingaffinity for the analyte of interest.

[0017] According to a first aspect of the present invention, anapparatus to determine the concentration of a certain metaboliteoccurring within a solution is provided. The apparatus comprises a teststrip which changes color in response to the binding of ananalyte-specific biological recognition molecule (e.g. an antibody, areceptor, a piece of DNA) or a portion of such a recognition moleculewhich retains relevant binding characteristics, and its binding partner.The color change results from response of a lipophilized chromophorewhich changes its color (or fluorescence) in response to theconcentration of the specific ion (such as H+). Such a molecule isreferred to as a “chromionophore (or fluorionophore)”. The ability ofthe chromionophore (or fluorionophore) to sense the true concentrationof the specific ion is modulated by the amount of binding of thebiological recognition molecule for its binding partner. The mechanismfor this modulation of chromionophore (or fluorionophore) response tothe binding between the biological recognition molecule and its bindingpartner can be direct or indirect as follows:

[0018] Direct modulation: The chromionophore (or fluorionophore) isattached chemically to a molecule having an affinity for the biologicalrecognition molecule. Such a molecule is hereafter referred to as a“chromaffinophore (or fluoraffinophore)”. When the recognition moleculebinds to the chromaffinophore (or fluoraffinophore), the chromionophore(or fluorionophore) portion is obstructed by the relatively largebiological recognition molecule. This alters the chromaffinophore (orfluoraffinophore)'s ability to interact chemically with the ion to whichit specifically responds.

[0019] Indirect modulation: The chromionophore (or fluorionophore)exists in an ion exchange context with a second ionophore which binds asecond ion and transports it into a lipophilic environment such as amembrane. Charge neutrality must be maintained within the membrane. Toachieve this, the membrane also contains a limited concentration of alipophilic ion of opposite charge to that of the ions which aretransported by the ionophore and chromionophore (or fluorionophore).Both the chromionophore (or fluorionophore) and the other ionophorecompete for charge neutralization by this counter-ion in carrying theirrespective ions into the membrane. In the case of indirect modulation,the ionophore is attached chemically to a molecule having an affinityfor the biological recognition molecule. Such a molecule is hereafterreferred to as an “affinophore”. The binding of the recognition moleculeto the affinophore alters its ability to transport its specific ion intothe membrane. This makes more counter-ion available to thechromionophore (or fluorionophore) thereby permitting increasedtransport of its ion into the membrane and causing a shift in the ionconcentration at which a color or fluorescence change occurs in thechromionophore (or fluorionophore).

[0020] Thus the test strip may be comprised in two possible combinationsof components, the first combination being:

[0021] 1a) an absorbent matrix through which the liquid containing theanalyte to be measured is carried to a membrane.

[0022] 2a) membrane affixed to and/or in contact with the matrix, saidmembrane containing

[0023] a) certain lipophilic ions, each of which has a certainelectrical charge;

[0024] b) a chromionophore (or fluorionophore) which is also adapted tocarry a first ionic species into said membrane where said ionic specieswill neutralize a portion of the certain electrical charge of a portionof said lipophilic ions, and which will change color (or fluorescence)when it carries an ion into the membrane.

[0025] c) A plurality of affinophores, each of which is adapted to carryinto said membrane a second ionic species which after entering themembrane will neutralize the certain electrical charge of a portion ofsaid lipophilic ions, and which possesses a binding affinity for arecognition molecule which binds to the substance being measured.

[0026] 3a) a plurality of recognition molecules which bind both to thesubstance to be measured and to the affinophore, said recognitionmolecule being present on the absorbent matrix and/or added to oralready present in the solution containing the analyte.

[0027] 4a) a source of ions which will be transported by thechromionophores (or fluorionophores) and affinophores, said ions beingcontained in the absorbent matrix and/or added to or already present inthe solution matrix, the concentration of said ions available to themembrane being either controlled (e.g. buffered) or in excess.

[0028] The second combination of components comprising the test stripsis

[0029] 1b) an absorbent matrix through which the liquid containing theanalyte to be measured is carried to a membrane.

[0030] 2b) membrane affixed to and/or in contact with the matrix, saidmembrane containing

[0031] a) certain lipophilic ions, each of which has a certainelectrical charge;

[0032] b) a plurality of chromaffinophores (or fluoraffinophores) eachof which is adapted to carry an ion or ions to said membrane, and whichwill change color (or fluorescence) when it carries an ion into themembrane, and which possess a binding affinity for a recognitionmolecule which binds to the analyte being measured.

[0033] 3b) a plurality of recognition molecules which bind to both theanalyte to be measured and to the chromaffinophore (orfluoraffinophore), said recognition molecules being present on theabsorbent matrix and/or added to or already present in the solutionbeing measured.

[0034] 4b) a source of ions which will be transported by thechromaffinophores (or fluoraffinophores) said ions being contained inthe absorbent matrix and/or added to or already present in the solutionmatrix, the concentration of said ions available to the membrane beingeither controlled (e.g. buffered) or in excess.

[0035] Either of the above embodiments may be modified to incorporatethe placement of a chemical marker at some point on the strip, saidchemical marker providing a visible change resulting from exposure ofthe marker to the liquid which carries the sample. This marker serves toalert the user that the solvent front carrying the sample has reachedthe point at which the strip should be read.

[0036] According to a second aspect of the present invention, a methodis provided to determine the concentration of a certain analyteoccurring within a solution. When used with the embodiment of theapparatus which does not possess the ionic species impregnated into theabsorbent matrix, the method comprises the following steps:

[0037] 1) adding a specified quantity of recognition molecules and/or ofionic species and/or buffering compounds to a specified volume ofsample.

[0038] 2) Mixing the sample

[0039] 3) Waiting a prescribed period of time greater than one (1)second and less than one (1) hour.

[0040] 4) Placing a test strip of the type described in the first aspectof invention into a volume of a sample which is to be tested for a timesufficient to allow the sample to permeate the absorbent matrix and/orto chromatograph into the matrix, so as to bring the sample andrecognition molecule into contact with the membrane, thereby allowingrecognition molecules which are not bound to the analyte in the sampleto bind to the affinophore or chromaffinophore (or fluoraffinophore) inthe strip molecules, thereby affecting the color or the membrane in amanner related to the concentration of said analyte within said medium.

[0041] 5) Stopping the flow of the sample into absorbant matrix eitherby removing the strip from the sample or by controlling the volume ofsample so that the supply is exhausted.

[0042] 6) Reading the test strip with the help of a calibrated scaleprovided with the test strip kit.

[0043] Additionally, according to a second aspect of the presentinvention, a second method is also provided to determine theconcentration of a certain analyte occurring within a solution. Thismethod will be used with the embodiment of the apparatus which possessesboth the ionic species and the recognition molecules impregnated intothe absorbent matrix. The method comprises the steps:

[0044] 1) placing a test strip of the type described in the first aspectof the present invention into a volume of a sample which is to be testedfor a time sufficient to allow the sample to permeate the absorbentmatrix and/or to chromatograph into the matrix, so as to bring thesample and recognition molecule into contact with the membrane, therebyallowing recognition molecules which are not bound to the analyte in thesample to bind to the affinophore or chromaffinophore (orfluoraffinophore) in the strip molecules which are not bound to theanalyte in the sample to bind to the affinophore or chromaffinophore (orfluoraffinophore) in the strip molecules, thereby affecting the color orthe membrane in a manner related to the concentration of said analytewithin said medium.

[0045] 2) Stopping the flow of the sample into absorbant matrix eitherby removing the strip from the sample or by controlling the volume ofsample so that the supply is exhausted.

[0046] 3) Reading the test strip with the help of a calibrated scaleprovided with the test strip kit.

[0047] Third and fourth embodiments of the second aspect of theinvention result in the case of strips which incorporate the placementof a chemical marker at some point on the strip, said chemical markerproviding a visible change resulting from exposure of the marker to theliquid which carries the sample. This marker (hereafter reffered to as“the end point marker”) serves to alert the user that the solvent frontcarrying the sample has reached the point at which the strip should beread. For these strips, step 1 of the procedures becomes:

[0048] 1) placing a test strip of the type described in the first aspectof the present invention into a volume of a sample which is to be testedfor a time sufficient to allow the sample to permeate the absorbentmatrix and/or to chromatograph into the matrix, so as to bring thesample and recognition molecule into contact with the membrane, therebyallowing recognition molecules which are not bound to the analyte in thesample to bind to the affinophore or chromaffinophore (orfluoraffinophore) in the strip molecules which are not bound to theanalyte in the sample to bind to the affinophore or chromaffinophore (orfluoraffinophore) in the strip molecules, thereby affecting the color orthe membrane in a manner related to the concentration of said analytewithin said medium; said sufficient time being defined by a change inthe end point marker.

[0049] Further objects, features, and advantages of the presentinvention will become apparent from a consideration of the followingdescription and the appended claims when taken in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] For a fuller and more complete understanding of the nature andobjects of the present invention, reference should now be made to thefollowing drawings in which:

[0051]FIG. 1 is a front view of an apparatus made in accordance with theteachings of the preferred birth management embodiment of the inventionand shown immersed within a medium. This embodies the indirectmodulation with the recognition molecule on the strip;

[0052]FIG. 2 is a top view of the apparatus shown in FIG. 1 illustratingthe use of color generation to illustrate the determined concentrationof metabolite within the medium of FIG. 1;

[0053]FIG. 3 is a front view of a birth management apparatus comprisingan embodiment of the invention utilizing direct modulation andrecognition molecule impregnated on the strip.

[0054]FIG. 4 is a front view of a birth management apparatus comprisingan embodiment of the invention utilizing indirect modulation with therecognition molecule in the solution.

[0055]FIG. 5 is a front view of a birth management apparatus comprisingan embodiment of the invention utilizing direct modulation with therecognition molecule in the solution.

[0056] FIGS. 6(a)-6(c) are top views (photographs) of birth managementapparatus comprising embodiments of the invention where 1) the membraneis applied to the strip over a continuous region and 2) where themembrane is applied to the strip in a series of dots, both arrangementsembodying the principles of the preferred embodiment of the invention;and

[0057]FIG. 7 is a chemical reaction schematic diagram illustrating thesynthesis of an affinophore utilized in the preferred embodiment of theinvention.

[0058]FIG. 8 shows the molecule, [2-dodecyl-2-methyl-1,3-propanediylbis[N-5′-amido-estrone-3-glucuronidyl-(benzo-15-crown-5)-4′-yl]carbamate]],the affinophore molecule for Eig utilized in the preferred embodiment ofthe invention.

DETAILED DESCRIPTION OF THE INVENTION

[0059] Referring now to FIG. 1, there is shown an apparatus 10 formeasurement of analyte concentration or presence, made in accordancewith the teachings of the present invention and useful for birthmanagement applications. It should be noted by those of ordinary skillin the art that the following discussion delineates methodologicalprinciples underlying and forming and integral part of Applicant'sinvention and that these principles may be used quite apart from thebirth management technique which is hereafter discussed. As shown,apparatus 10 includes, on one side, a generally absorbent matrix 12,such as commercially available filter paper, onto which is placed agenerally lipophilic membrane 14. In the preferred embodiment of thepresent birth management invention, the lipophilic membrane matrix 14comprises such as and without limitation, a mixture of polyvinylchloride, polyurethane and bis(2-ethylhexyl) sebacate and contains aplurality of chromionophores 18, such as and without limitation ETH 5350each of which may be bound to a positively charged ion 20 such as andwithout limitation, hydrogen (H+), and capable of carrying the bound ion20 from the interface between the membrane and the liquid permeating theabsorbent matrix 12, into the interior of the membrane 14, and whichchanges color when bound to said ion. Said membrane also contains aplurality of affinophores 22, such as and without limitation,[2-dodecyl-2-methyl-1,3-propanediylbis[N-5′-amido-estrone-3-glucuronidyl-(benzo-15-crown-5)-4′-yl]carbamate],each of which may be bound to a positively charged ion 24 such as, andwithout limitation, potassium-(K+), and capable of carrying said boundion 24 from the interface between the membrane and the liquid in theabsorbent matrix 12, into the interior of the membrane 14. Said membranealso contains a plurality of lipophilic anions 16, such as and withoutlimitation, sodium tetrakis [3,5-bis(trifluoromethyl)phenyl]borate, theconcentration of said anions being limited so that maintainance ofcharge neutrality within the membrane requires competition betweenchromionophores 18 and affinophores 22.

[0060] Also in the preferred embodiment of the present invention, aplurality of recognition molecules 42, such as and without limitation,anti-estrone-3-glucuronide antibody, are deposited on the absorbentmatrix 12 at a location between the membrane and the end of theabsorbent matrix 12 which is to make contact with the sample. In analternative embodiment, the plurality of recognition molecules 42 aredeposited on the area of the absorbent matrix which contacts themembrane 14.

[0061] As should be apparent to one of ordinary skill in the art,chromionophores 18 exhibit a first color, such as and withoutlimitation, the color “blue” when carrying a positively charged ion suchas and without limitation, H+ ion 20, and a second and different color,such as and without limitation, the color “pink” when not carrying thispositively charged ion. Moreover, affinophores 22 for use in Applicant'stechniques may be synthesized from commercially available ionophoresthrough chemical and/or biological alteration or conjugation with or toa molecule which may comprise a binding molecule, such as and withoutlimitation, an antigen identical to or resembling the analyte whoseconcentration is to be measured using an antibody as a biologicalrecognition molecule, such as and without limitationestrone-3-glucuronide. Affinophores 22 may also be synthesized which arebased upon ionophores which are not commercially available. Thebiological recognition molecules 42 may alternatively comprise anucleotide sequence having a chemical and/or biological binding affinityfor the analyte, a biological receptor for the analyte, an amino acidsequence which confers binding for the analyte, or virtually anysubstance which has some chemical and/or biological binding affinity forthe analyte of interest. Upon binding to the biological recognitionmolecule, the affinophore's ability to bind and carry ions into themembrane is reduced and/or compromised and/or eliminated. It first mustbe realized that charge neutrality must be maintained within themembrane. Thus, a reduction in the number of charged ions carried by theaffinophore must necessarily be compensated for by a concomitantincrease in the number of charged ions carried into the membrane by thechromionophore. Hence, the color of the membrane is shifted toward thecolor characteristic of the charge carrying chromionophore.

[0062] In the preferred birth management embodiment of Applicant'spresent invention, affinophore 22 comprises[2-dodecyl-2-methyl-1,3-propanediylbis(N-5′-amido-estrone-3-glucuronidyl-(benzo-15-crown-5)-4′-yl]carbamate].This is shown in FIG. 8. Particularly, in one embodiment of the birthmanagement embodiment, the affinophore was synthesized by Applicants inthe following manner and shown schematically in FIG. 7:

[0063] (1) Synthesis of Methyl[17-Oxaestra-1,3,5(10)-trien-3-yl-2′,3′,4′-tri-O-acetyl-D-glucopyranosid]Uronate (3):

[0064] Toluene (300 ml) was added to a flame dried 500 ml three neckedflask containing estrone 2 (1.67 g, 6.18 mmol) and cadmium carbonate(2.13 g, 12.36 mmol). A short path distillation assembly with a receiverwas attached to one of the necks and the whole set up was kept underinert atmosphere. Toluene was distilled (25 ml) to ensure dryness to thecontents of the flask. In another flame dried flask (100 ml) was takenbromo sugar 1 (4.91 g, 12.36 mmol) in toluene (90 ml). This solution wasadded dropwise to a stirred mixture of estrone and cadmium carbonate for1 h and an equal volume of toluene distilled from the flask at roughlythe same rate. After the addition was complete, the distillationassembly was replaced by a reflux condenser and the mixture refluxed for4 days. The mixture was filtered as a hot solution over a pad of celite,and the filtrate was evaporated to an oil. The oil obtained wascrystallized by dissolving it in CH₂Cl₂ and adding absolute ethanol tothe boiling solution. The resulting crystals were recrystallized fromCH₂Cl₂-EtOH to obtain 3 as colorless crystals (2.33 g, 64%). Analyticalmaterial was obtained by further purification by flash chromatography[EtOAc-Hexane, 40-60% EtOAc]. Rf 0.56 (EtOAc:Hexane, 6:4).

[0065] IR (Kbr): 1758, 1498, 1226, 1100, 1053 cm-1.

[0066] 1H NMR (CDCl₃): 0.9 (s, 3H, CH₃), 2.03 (s, 3H, OCOCH₃), 2.04, (s,3H, OCOCH₃), 2.05 (s, 3H, OCOCH₃), 2.88 (m, 2H), 3.74 (s, 3H, COOCH₃),4.16 (m, 1H, H-5), 5.09 (s, 1H), 5.11 (s, 1H), 5.32 (m, 4H), 6.73 (d,J=2.4 Hz, 1H), 6.78, 6.79 (dd, J=8.4 Hz, 2H), 7.21 (d, J=8.4 Hz, 1H).

[0067] (2) Hydrolysis of Acetylated Glycosides. 17-Oxoestra-1,3,5(10)-trien-3-yl-β-D-glucopyranosid Uronic Acid (5):

[0068] To a suspension of 3 (0.1 g, 0.177 mmol) in CH₂Cl₂ (2 ml) wasadded Ba(OH)₂. 8H₂O (0.03 g, 0.097 mmol) in methanol (1.95 ml) added asa stock solution and let stir for 0.5 h. TLC shows complete conversionof the starting material. The solvents were evaporated and the residuedissolved in a mixture of EtOAc, −0.05N HCl-saturated brine solution(1:1, 20 ml) and stirred for 5-10 minutes. The contents were transferredto a separatory funnel and the layers separated. The organic layers werewashed with brine, dried over anhydrous MgSO4, filtered and concentratedto afford 5 as a flaky substance (0.89 g).

[0069] IR (KBr): 3422, 1727, 1498, 1245, 1086, 1033 cm-1.

[0070] (3) Synthesis of 4′-[N-Dodecanoylamino]benzo-15-crown-5 (8):

[0071] Dodecanoyl chloride 7 (1.7 g, 7.79 mmol, 1.8 ml) in 20 mltetrahydrofuran (THF) was added dropwise to an anhydrous THF solution(40 ml) containing 4′-aminobenzo-15-crown-5 6 (2 g, 7.08 mmol) andtriethylamine (1.58 g, 15.58 mmol, 2.17 ml) at room temperature andunder nitrogen atmosphere. After the reaction was complete (2 h), theprecipitate was filtered, and the filtrate concentrated to dryness invacuo. The residue was dissolved in chloroform (75 ml), washed withwater (25 ml), dried over MgSO₄, filtered and concentrated. The crudesolid obtained was recrystallized from ethanol to obtain 1.5 g ofcolorless crystals. The filtered precipitate was dissolved in water (25ml) and chloroform (75 ml) and extracted. The organic layers were washedwith water (25 ml) and brine (25 ml), dried over MgSO4, filtered andconcentrated to obtain a solid (1.51 g) which corresponded to the purerecrystallized product 8 by TLC and 1H NMR. Both of the products werecombined to obtain 8 (3.01 g, 91% yield).

[0072] IR (KBr): 3286, 1656, 1516, 1241, 1141 cm-1.

[0073] 1H NMR (CDCl₃): 0.87 (t, J=6.5, 7.5 Hz, 3H), 1.25 (m, 16H), 1.7(m, 2H), 2.31 (t, J=7.5 Hz, 2H), 3.75 (d, J=3 Hz, 8H), 3.88 (q, 4H),4.12 (m, 4H), 6.8 (d, J=1.5 Hz, 2H), 7.12 (s, 1H, NH), 7.38 (d, J=1.5Hz, 1H).

[0074] (4) Synthesis of 4′-[N-Dodecylamino]benzo-15-crown-5 (9)

[0075] Compound 8 (2.9 g, 6.29 mmol) in anhydrous THF (90 ml) wastreated with lithium aluminum hydride (0.72 g, 18.87 mmol) and refluxedfor 4 h. After the reaction mixture was cooled in an ice bath, excessLiAlH₄ was carefully decomposed in chloroform (75 ml), washed with water(20 ml), brine (20 ml), dried over MgSO₄, filtered and concentrated. TLCand 1H NMR show presence of no other products or impurities and thecompound (9) obtained was used as such. [2.86 g, quantitative] Rf 0.5(EtOH:CHC13, 6:94).

[0076] IR (KBr): 1518, 1230, 1130, 1106, cm-1.

[0077] 1H NMR (CDCl₃): 0.87 (t, J=6.6, 6.9 Hz, 3H), 1.26 (s, 18H), 1.59(m, 2H), 3.03 (t, J=7.2, 6.6 Hz, 2H), 3.75 (s, 8H), 3.88 (m, 4H), 4.08(m, 4H), 6.13 (dd, J=8.7, 8.4 Hz, 1H), 6.21 (d, J=2.7 Hz, 1H), 6.77 (d,J=8.4 Hz, 1H).

[0078] (5) Coupling reaction between 5 and 9 with BOP-CI (10):

[0079] In a flame dried (100 ml) flask were taken 5 (0.89 g, 0.19 mmol),9 (0.07 g, 0.15 mmol) and triethylamine (0.03 g, 0.04 ml) in anhydrousTHF (20 ml). In another flask was taken BOP-C1 10 (0.3 g) dissolved in amixture of anhydrous THF (16 ml) and CH2C12 (5 ml). From this, analiquot (2.8 ml, 0.15 mmol) was taken and added dropwise to the aboveflask containing compounds 5 and 9 at room temperature and left stirringovernight (˜10 h). Water (10 ml) was added and stirred for 5 minutes andthe solvents were evaporated under vacuum. EtOAc (50 ml) was added andthe layers separated, the organic layers were washed with brine (20 ml),dried over MgSO₄, filtered and concentrated. The residue obtained waspurified by gravity column using silica gel (100-200 mesh) and elutingwith a gradient of EtOH-CHCl₃ (2-8% EtOH). Unreacted4′-[N-dodecylamino]benzo-15-crown-5 (9) (0.055 g) followed by the pureadduct 11 (0.015 g, 51% based on the consumption of 8. Rf 0.24(EtOH:CHCl₃, 6:94).

[0080] IR (KBr): 3385, 1736, 1514, 1265, 1128, 1056 cm-1.

[0081] 1H NMR (CDCl₃) 0.88 (t, 3H, terminal CH₃), 0.92 (s, 3H, c-13CH₃), 1.25 (s, alkyl chain), 3.74 (s, 8H), 3.86 (m, 4H), 4.07 (m, 4H),6.68, 6.71, 6.79, 6.85, 7.19 (aromatic).

[0082] In the synthesis reported above, the appearance of the peak inthe IR at 1265 suggests that side reaction has occurred between BOP-CIand the hydroxyls of the steroid glucuronide. BOP-CI was used in thepreviously described method because coupling was occurring between asecondary amine and a carboxyl group. By changing the starting materialso that a primary amine is used, it becomes possible to utilizedecyclohexylcarbodiimide in place of BOP-CI as the coupling agent. It isexpected that this will eliminate the suspected side reaction. Thesynthesis will instead proceed from 4′-amino-5′-nitrobenzo-15-crown-5.The amine will be reacted with dodecanoyl chloride, placing the dodecylnoiety at the 4′ nitrogen. The amide and the 5′-nitro group could thenbe reduced in a single step with either sodium borohydride and titaniumtetrachloride or sodium borohydride and cobalt chloride hexahydrate toafford 4′-dodecylamino-5′-aminobenzo-15-crown-5. If the single stepreduction of both the nitro and amide is not successful, they could bereduced step wise with lithium aluminumhydride for the amide groupfollowed by the reaction of nitro group with 10% palladium on carbonunder hydrogen. The coupling to the steroid glucuronide will thenproceed using dicyclohexylcarbodiimide as the coupling agent rather thanBOP-CI.

[0083] It should be realized by one of ordinary skill in the art thatwhile the aforementioned affinophores was employed in this birthmanagement embodiment, other types of affinophores may be utilized. Ingeneral, an affinophore for use in Applicants' techniques as earlierstated are entities having a binding affinity for the metabolite ofinterest and the ability to carry an ion into a lipophilic membrane.

[0084] Referring now to FIGS. 6(a)-6(c), there are shown strip testsmade in accordance with the principles of the preferred embodiment ofthe present invention. Specifically, the test strip 10 comprises a stripof Whatman chromatography paper grade 20 which had dimensions of about 4mm×10 cm in this embodiment, about 3 μl of membrane solution was spreadon the surface of the strip in an area occurring between about 2.5 cm toabout 4.0 cm from the bottom of the strip. About 1.0 μl of antibodysolution was placed in a stripe upon the strip matrix at a point about 2cm from the bottom of the strip.

[0085] Moreover, in the preferred birth management embodiment themembrane solution was prepared which utilized chemicals (obtained fromFluka, Ronkonkoma, N.Y.) in the following amounts: 200 μl of a solutioncomprising about 20 mg of polyvinyl chloride (high molecular weight), 40mg of bis(2-ethylhexyl) sebacate and about 1 ml of redistilledtetrahydrofuran, about 0.0038 mmole of ETH 5350 chromionophore III perkg of PVC/DOS (1:2) and an amount of the lipophilic anion, sodiumtetrakis [3,5-bis(trifluoromethyl)phenyl]borate, and the aforedescribedaffinophore, sufficient to create a substance ratio of about 1:1.1:10 ofchromionophore to anion to affinophore. (This formulation if referred toas batch 1 in FIG. 6). A second formulation referred to as batch 2comprises the following: 200 μl PVC/DOS (20 mg PVC+40 mg DOS in 1 ml.THF), 10 μl of sodium tetrakis [3,5-bis(trifluoromethyl)phenyl]borate(1.67 mg/ml of THF and 100 μl of ETH 5350 chromionophore (orfluorionophore) III (1.167 mg/ml of THF and 100 μl of the affinophore (4mg/200 μl THF).

[0086] The antibody was anti-estrone-3-glucuronide monclonal antibodyclone #278-17144 which was obtained by and through OEM Concepts, Inc. ofToms River, N.J. The solution contained about 2×10⁻⁹ moles of antibodyin about 1.0 μl of 0.1M bis-tris propane buffer having a pH of about7.002.

[0087] When all of the solutions had dried on the strip, the strip wasmounted vertically so that its bottom touched the bottom of a well of anELISA plate, the strip being held perpendicular to the well. The stripdid not contact any other surface below the mounting point. About 40 μlof 0.1M bis-tris propane buffer having a pH of about 7.002 was placedinto the well and allowed to rise up the strip chromatographically,distributing the antibody over the surface of the membrane where it isbound by the affinophore. About 10 μl of the sample to be measured wasadded to about 1 ml of about 0.1M KCl in about 0.1M bis-tris propanebuffer, having a pH of about 7.0. About 20 μl of this solution waschromatographed up the strip and the height at which the strip changedfrom blue to pink correlated with the concentration ofestrone-3-glucuronide in the sample. Example strips demonstrating about20 ng and about 80 ng of estrone-3-glucuronide samples and also tests onactual urine samples collected during the ovulatory and non-ovulatoryphases are shown in FIGS. 6(a)-6(c).

[0088] In order for apparatus 10 to reliably indicate the concentrationof the desired metabolite within medium 30, the ion concentration withinthe medium must be controlled. Two control methods include, withoutlimitation, maintaining the ions present in sufficient excess over theamounts of affinophore and chromaffinophore (or fluoraffinophore) in theapparatus 10 so that variations in the actual concentrations of the ionsin the medium produce substantially no significant change in the amountof the ions transported to and within membrane 14 and/or buffering theion concentrations in the medium by the use of commercially available pHor metal buffers so as to insure that the available free ions within thesolution remain substantially constant.

[0089] As shown in FIGS. 1 and 2, one end of apparatus 10 is adapted tobe immersed within medium 30 containing analyte 40. In operation, theaffinophores 22 compete with the chromionophores (or fluorionophores) 18for a limited amount of counterion 16. Upon entry into the membrane,ions 20 and 24, respectively carried into membrane 14 by chromionophores(or fluorionophores) 18 and affinophores 22, neutralize correspondingcounterions 16. Since the membrane 14 must remain electrically neutral,the amount of counter-ions 16 determines the overall amount ofaffinophores 22 and chromionophores (or fluorionophores) 18 which maycarry ions 20 and 24 into the membrane 14 at any instant in time. Hence,there is a competition between affinophores 22 and chromionophores (orfluorionophores) 18 for entry of their respective ions into themembrane. However, the use of biological recognition molecules 42“skews” this competition in favor of the chromionophores (orfluorionophores) 18, by an amount which is proportional to theconcentration of metabolite 40 occurring within the medium 30. Inessence, a first substance (ions 20 carried by chromionophores (orfluorionophores) 18) competes with a second substance (ions 24 carriedby affinophores 22) for entry into the membrane 14 in order toneutralize the charge on the plurality of counter-ions 16. Thebiological recognition molecules 42 (third substance) modulates thiscompetition by attaching to and/or binding with a portion of theaffinophores 22, thereby preventing the bound affinophores (such asaffinophore 26 which is shown in FIG. 1) from carrying an ion intomembrane 14. In this manner, more of the counter-ions 16 are availablefor the chromionophores (or fluorionophores) 18 to neutralize. Hence,more ion containing chromionophores (or fluorionophores) 18 enter themembrane 14 than would enter without the presence of the biologicalrecognition molecules 42, thereby causing a shift in the ionconcentration at which a color change occurs in the chromionophores (orfluorionophores) 18. A mathematical description of these relationshipsis set forth in the set of equations found in the article entitled“Selectivity of Ion-Sensitive Bulk Optodes”, which was authored by E.Bakker and W. Simon, which appeared in Volume 64 on pages 1805-1812 ofthe journal entitled Analytical Chemistry, published in 1992, which isfully and completely incorporated herein by reference, word for word andparagraph for paragraph. In practice, the color of the matrix 12 willcorrespond to that of the chromionophore (or fluorionophore) whenL_(tot) (as defined in the aforementioned article in AnalyticalChemistry to be the concentration of ion-selective ionophore in themembrane) is at a maximum. These biological recognition molecules 42bind first to the metabolite 40 occurring in the medium, with theremainder binding to the affinophores 22. In this manner, one maycalibrate the increase in chromionophore (or fluorionophore) entry intomembrane 14 since it is proportional to the amount of metabolites 40existing within the solution medium 30. Moreover, the amount ofbiological recognition molecules attached to the various affinophores 22is inversely proportional to the amount of metabolites 40 existing oroccurring within the medium 30.

[0090] When the end of apparatus 10 is placed into the medium 30,antibody of biological recognition molecules 42, which have not beenbound to the analyte molecules in the sample 30 will be bound to theaffinophores 22, in the manner previously discussed, thereby allowingmore chromaffinophores (or fluoraffinophores) 18 to carry ions 20 intothe membrane 14. In the preferred embodiment of the present invention,the bottom of apparatus 10 will be placed in an amount of diluent sampleliquid sufficient to rise chromatographically up the strip to apredetermined level. As the chromionophore (or fluorionophore) entersthe membrane 14, color changes occur along the matrix 12 (as shown inFIG. 2). At some point along the strip, a sufficient amount of antibody42 will have been bound so the amount remaining which binds to theregion of the strip is not sufficient to cause the strip to changecolor. This point will divide strip matrix 12 into two regions 60, 70.The amount of antigen or metabolite which causes this to occur atvarious points 80 along strip matrix 12 will be calibrated so that theconcentration can be read, in a relatively easy manner, by the divisionpoint along the strip 12. Hence, the strip test of this invention or“home-based” testing technique utilizes a distinct transition from onecolor to another, such as commonly seen with pH indicators, as thevisual signal must be recognized. Quantification of the response will beaccomplished spatially in a manner similar to that of a thermometer. Theindividual reading of the strip will simply compare the location alongthe strip where the color change occurs with a calibration scale (suchas 80) on the side of the strip 12. The method of this embodiment of thepresent invention will require no “rinse” steps and the components ofthe strip matrix 12 are considerably more stable than enzymes which areused in prior techniques.

[0091] In a second embodiment of the present birth management invention,shown in FIG. 3, the chromionophore (or fluorionophore) 19 is chemicallyand/or biologically attached to a molecule having a binding affinity forthe biological recognition molecule, the molecule conjugate hereinafterreferred to as a “chromaffinophore (or fluoraffinophore)”. In thisembodiment, there is no competing ionophore or affinophore. When therecognition molecule 42 binds to the chromaffinophore (orfluoraffinophore) 19, the chromaffinophore (or fluoraffinophore) portionis obstructed by the relatively large biological recognition molecule(as shown by 26). This alters the chromaffinophore (orfluoraffinophore)'s ability to chemically interact with the ion 16 towhich it responds. Hence, the amount of binding of the recognitionmolecule to the chromaffinophore (or fluoraffinophore) is inverselyproportional to the amount of metabolite within the medium 30. Moreover,it should be appreciated by one of ordinary skill in the art that eachof the embodiments may be used to simply change color (without theaforedescribed calibration strip portion) to indicate the presence ofthe metabolite, and that various types of metabolites may be detected inpresence or concentration, including and without limitation,pregnanediol-glucuronide, estrone 3-glucuronide and estriol glucuronide.

[0092] Moreover, in third and fourth embodiments of the presentinvention, the recognition molecule is placed in the sample rather thanon the test strip, in each of these embodiments which, in all otherways, respectively correspond to the first and second embodiments of theinvention (as sown in FIGS. 4 and 5).

[0093] In a fifth variation of the preferred embodiment of the presentinvention, the affinophore test strip placed the membrane solution in aseries of about 1 micro-liter dots which were spaced at about 2.5, 3.25,and about 4 cm from the bottom of the strip. This facilitatesvisualization in terms of the number of dots that have become pinkrather than a continuous field with a demarcation line between blue andpink. All other procedures remained the same. This is shown in FIG. 6,examples b and c.

[0094] It is to be understood that the invention is not limited to theexact construction and modifications illustrated and described above,but that various changes and modifications may be made without departingfrom the spirit and the scope of the invention as defined in thefollowing claims.

[0095] It should be realized by one of ordinary skill in the art thatwhat has been disclosed is one embodiment of a novel technique which isa new and useful type of visual immunoassay, one based uponaffinophores, which obviates the need for prior costly techniques suchas radioisotope type immunoassays or enzyme linked antibody assays.Moreover, it should be further apparent that the aforedescribed birthmanagement invention is both relatively inexpensive and relatively easyto use, thereby having great utility as a “home-based” birth managementsystem and as a veterinary means of monitoring animal fertility. Itshould also be realized that the use of such affinophore binding allowsconcentrations of metabolites to be sensed in much smaller concentrationamounts than previously possible with “home-based” techniques.

In the claims:
 1. A test apparatus comprising an absorbent matrix ontowhich is placed in intimate contact a lipophilic membrane solutioncontaining a plurality of molecules from the groups of chemicalscomprising: a chromionophore , an affinophore, and a lipophilic counterion; and onto which is also placed a plurality of biological recognitionmolecules which bind to the analyte in the sample, and have a bindingaffinity for the affinophore; said test apparatus also comprising anadditive mixture containinging: buffering substances, ions having anaffinity for the chromionophore and ions having an affinity for theaffinophore, said absorbant matrix being adapted to be placed within asample medium of composition comprising: sample analyte to be measuredand said additive mixture.
 2. The test apparatus of claim 1 wherein saidbiological recognition molecule comprises an antibody.
 3. The testapparatus of claim 1 wherein said biological recognition moleculecomprises a portion of an antibody.
 4. The test apparatus of claim 1wherein said biological recognition molecule is a biological receptorfor the analyte.
 5. The test apparatus of claim 1 wherein saidbiological recognition molecule is a portion of a specific nucleotidesequence having an affinity for the analyte.
 6. The test apparatus ofclaim 1 wherein said analyte comprises estrone-3-glucuronide.
 7. Thetest apparatus of claim 1 wherein said analyte comprisespregnanediol-glucuronide.
 8. The test apparatus of claim 1 wherein saidanalyte comprises estriol-glucuronide.
 9. The test apparatus of claim 1wherein the ion having an affinity for the chromionophore comprises H+,the ion having affinity for the affinophore is K⁺, the chromionophore is9-(diethylamino)-5-[(2-octyldecyl)imino]-5H-benzo[a]phenoxazine (knownto those skilled in the art as ETH 5350), the lipophilic anion is sodiumtetrakis [3,5-bis(trifluoromethyl)phenyl]borate, the affinophore is[2-dodecyl-2-methyl-1,3-propanediylbis[N-5′-amido-estrone-3-glucuronidyl-(benzo-15-crown-5)-4′-yl]carbamate]and the analyte is estrone-3-glucuronide.
 10. A test apparatuscomprising an absorbent matrix onto which is placed in intimate contacta lipophilic membrane solution containing a plurality of molecules fromthe groups of chemicals comprising: a chromionophore, an affinophore,and a lipophilic counter ion; said test apparatus also comprising anadditive mixture containing: buffering substances, ions having anaffinity for the chromionophore, ions having an affinity for theaffinophore, and a plurality of biological recognition molecules whichbind to the analyte in the sample, and have a binding affinity for theaffinophore; said absorbant matrix being adapted to be placed within asample medium of composition comprising: sample analyte to be measuredand said additive mixture.
 11. The test apparatus of claim 10 whereinsaid biological recognition molecule comprises an antibody.
 12. The testapparatus of claim 10 wherein said biological recognition moleculecomprises a portion of an antibody.
 13. The test apparatus of claim 10wherein said biological recognition molecule is a biological receptorfor the analyte.
 14. The test apparatus of claim 10 wherein saidbiological recognition molecule is a portion of a specific nucleotidesequence having an affinity for the analyte.
 15. The test apparatus ofclaim 10 wherein said analyte comprises estrone-3-glucuronide.
 16. Thetest apparatus of claim 10 wherein said analyte comprisespregnanediol-glucuronide.
 17. The test apparatus of claim 10 whereinsaid analyte comprises estriol-glucuronide.
 18. The test apparatus ofclaim 10 wherein the ion having an affinity for the chromionophorecomprises H+, the ion having affinity for the affinophore is K⁺, thechromionophore is9-(diethylamino)-5-[(2-octyldecyl)imino]-5H-benzo[a]phenoxazine (knownto those skilled in the art as ETH 5350), the lipophilic anion is sodiumtetrakis [3,5-bis(trifluoromethyl)phenyl]borate, the affinophore is[2-dodecyl-2-methyl-1,3-propanediylbis[N-5′-amido-estrone-3-glucuronidyl-(benzo-15-crown-5)-4′-yl]carbamate]and the analyte is estrone-3-glucuronide.
 19. A test apparatuscomprising an absorbent matrix onto which is placed in intimate contacta lipophilic membrane solution containing a plurality of molecules fromthe groups of chemicals comprising: a chromaffinophore and a lipophiliccounter ion; and onto which is also placed a plurality of biologicalrecognition molecules which bind to the analyte in the sample, and havea binding affinity for the chromaffinophore; said test apparatus alsocomprising an additive mixture containinging: buffering substances andions having an affinity for the chromaffinophore, said absorbant matrixbeing adapted to be placed within a sample medium of compositioncomprising: sample analyte to be measured and said additive mixture. 20.The test apparatus of claim 19 wherein said biological recognitionmolecule comprises an antibody.
 21. The test apparatus of claim 19wherein said biological recognition molecule comprises a portion of anantibody.
 22. The test apparatus of claim 19 wherein said biologicalrecognition molecule is a biological receptor for the analyte.
 23. Thetest apparatus of claim 19 wherein said biological recognition moleculeis a portion of a specific nucleotide sequence having an affinity forthe analyte.
 24. The test apparatus of claim 19 wherein said analytecomprises estrone-3-glucuronide.
 25. The test apparatus of claim 19wherein said analyte comprises pregnanediol-glucuronide.
 26. The testapparatus of claim 19 wherein said analyte comprisesestriol-glucuronide.
 27. The test apparatus of claim 19 wherein the ionhaving an affinity for the chromionophore comprises H+, the ion havingaffinity for the affinophore is K⁺, the chromionophore is9-(diethylamino)-5-[(2-octyldecyl)imino]-5H-benzo[a]phenoxazine (knownto those skilled in the art as ETH 5350), the lipophilic anion is sodiumtetrakis [3,5-bis(trifluoromethyl)phenyl]borate, the affinophore is[2-dodecyl-2-methyl-1,3-propanediylbis[N-5′-amido-estrone-3-glucuronidyl-(benzo-15-crown-5)-4′-yl]carbamate]and the analyte is estrone-3-glucuronide.
 28. A test apparatuscomprising an absorbent matrix onto which is placed in intimate contacta lipophilic membrane solution containing a plurality of molecules fromthe groups of chemicals comprising: a chromaffinophore and a lipophiliccounter ion; said test apparatus also comprising an additive mixturecontaining: buffering substances, ions having an affinity for thechromaffinophore, and a plurality of biological recognition moleculeswhich bind to the analyte in the sample, and have a binding affinity forthe chromaffinophore; said absorbant matrix being adapted to be placedwithin a sample medium of composition comprising: sample analyte to bemeasured and said additive mixture.
 29. The test apparatus of claim 28wherein the ion comprises hydrogen.
 30. The test apparatus of claim 28wherein said biological recognition molecule comprises an antibody. 31.The test apparatus of claim 28 wherein said biological recognitionmolecule comprises a portion of an antibody.
 32. The test apparatus ofclaim 28 wherein said biological recognition molecule comprises abiological receptor for said analyte.
 33. The test apparatus of claim 28wherein said biological recognition molecule comprises a portion of abiological receptor for said analyte.
 34. The test apparatus of claim 28wherein said biological recognition molecule comprises a portion of aspecific nucleotide sequence having affinity for said analyte.
 35. Thetest apparatus of claim 28 wherein said analyte comprisesestrone-3-glucuronide.
 36. The test apparatus of claim 28 wherein saidanalyte comprises pregnanediol-glucuronide.
 37. The test apparatus ofclaim 28 wherein said analyte comprises estriol-glucuronide.
 38. Thetest apparatus of claim 28 wherein the ion having an affinity for thechromionophore comprises H+, the ion having affinity for the affinophoreis K⁺, the chromionophore is9-(diethylamino)-5-[(2-octyldecyl)imino]-5H-benzo[a]phenoxazine (knownto those skilled in the art as ETH 5350), the lipophilic anion is sodiumtetrakis [3,5-bis(trifluoromethyl)phenyl]borate, the affinophore is[2-dodecyl-2-methyl-1,3-propanediylbis[N-5′-amido-estrone-3-glucuronidyl-(benzo-15-crown-5)-4′-yl]carbamate]and the analyte is estrone-3-glucuronide.
 39. A method ofsemi-qualification in which the point of transition between two colorregions on a test matrix is compared with a calibration scale whichcorrelates the position of the transition with the concentration of theanalyte.
 40. An apparatus to determine the concentration of a certainmetabolite occurring within a solution comprising: (a) A membranecontaining a plurality of certain ions, each having a certain electricalcharge; (b) A first substance which is adapted to carry into saidmembrane an ion which balances said certain electrical charge of aportion of said plurality of said certain ions; (c) A second substancewhich is adapted to carry into said membrane a second ionic specieswhich balances said certain electrical charge of a portion of saidplurality of said certain ions; and (d) A third substance which bindswith a portion of said first substance, said portion of said firstsubstance being of an amount proportional to said concentration of saidmetabolite occurring within said solution.
 41. The apparatus of claim 40wherein said metabolite comprises estrone-3-glucuronide.
 42. Theapparatus of claim 40 wherein said first substance comprises anaffinophore.
 43. The apparatus of claim 40 wherein said second substancecomprises a chromionophore (or fluorionophore).
 44. The apparatus ofclaim 40 wherein said third substance comprises an antibody.
 45. Anapparatus for determining the concentration of a certain metaboliteoccurring within a solution, said apparatus comprising; (a) Color means,in contact with said solution, for producing a certain color evidencingsaid concentration of said certain metabolite; and (b) Binding means forbinding with a portion of said metabolite and for allowing only aportion of said binding means to bind said color means, thereby allowingsaid color to evidence said concentration of said certain metabolitewithin said solution.
 46. An apparatus of claim 45 wherein the colorevidence comprises the position on a matrix where an area of one coloris next to an area of a different color, said position being calibratedto a standard relating position of color change to concentration ofanalyte in the sample.
 47. An apparatus of claim 45 wherein the colorevidence comprises a series of spots which change from one color toanother in response to sample traveling through an absorbent matrix, thenumber of spots attaining a change from the original color beingcalibrated to the concentration of analyte in the sample.
 48. Theapparatus of claim 45 wherein said metabolite comprisesestrone-3-glucuronide.
 49. The apparatus of claim 45 wherein saidbinding means comprises an antibody.
 50. A method to determine theconcentration of a certain metabolite occurring within a solution, saidmethod comprising the steps of: (a) Placing a plurality of ions, eachhaving a certain electrical charge, within a membrane (b) Providing afirst substance which is adapted to carry into said membrane an ionicspecies which balances said certain electrical charge of a first portionof said plurality of ions; (c) Altering said first substance, effectiveto allow said altered first substance to bind to a biologicalrecognition molecule; (d) Providing a second substance which is capableof carrying into said membrane a second ionic species which balancessaid certain electrical charge of a second portion of said plurality ofions; and (e) Providing a third substance which binds to said biologicalrecognition molecule, thereby preventing a portion of said firstsubstance from entering said membrane, said portion of said firstsubstance being proportional to said amount of said metabolite occurringwithin said solution.
 51. The method of claim 50 wherein said metabolitecomprises estrone-3-glucuronide.
 52. The molecule[2-dodecyl-2-methyl-1,3-propanediylbis[N-5′-amidoestrone-3-glucuronidyl-(benzo-15-crown-5)-4′-yl]carbamate]]shown in FIG. 6, which may be used as an affinophore forestrone-3-glucuronide assay in any instrumental context requiring suchan affinophore including without limitation a strip test as described inthis application, an affinophore based fiber optic sensor forestrone3-glucuoronide and/or an affinophore based electrode forestrone-3-glucuronide.